Radiation therapy (RT) is an integral therapeutic modality for treating non-small cell lung cancer (NSCLC). However, lung cancer is often refractory to RT and molecular mechanisms mediating treatment resistance and tumor repopulation remain poorly defined. We have determined that the receptor tyrosine kinase EphA5 is highly expressed in lung cancer and, more importantly, its expression in patients negatively correlates with RT success and survival, thus suggesting its involvement in the regulation of cellular responses to genotoxic insult. We have assembled multiple lines of evidence to support a potential mechanism underlying EphA5-mediated radioresistance: (i) EphA5-silenced lung cancer cells display a defective G1/S cell cycle checkpoint and are unable to resolve DNA damage, (ii) upon irradiation, EphA5 is found in the nucleus of cells where it interacts with activated ataxia-telangiectasia mutated (ATM) at sites of DNA repair and, (iii) we demonstrated that a new monoclonal antibody against EphA5 sensitizes lung cancer cells and human lung cancer xenografts to RT, and significantly prolongs survival of tumor-bearing mice. In order to initiate the translation of our findings, we have developed a new methodology that utilizes in vitro and in vivo screening approaches based on phage and yeast antibody display to select and characterize antibodies with radiosensitizing properties, and capable of recognizing targets in vivo. In Aim 1, we will apply our combined hierarchical approach to generate anti-EphA5 antibodies with radiosensitizing properties and capable of targeting EphA5-expressing tumors in vivo. Aim 2 will study functions of EphA5 in DNA damage response, and how to prevent it for therapeutic purposes. Aim 3 will define biodistribution properties of selected antibodies. Toxicology studies, and exploratory pharmacodynamics/pharmacokinetics will also be performed. In Aim 4, we will test whether targeted inhibition of EphA5 combined with ionizing radiation will improve treatment outcomes.